Neonatal sunburn and melanoma in mice.

Retrospective epidemiological data have indicated that cutaneous malignant melanoma may arise as a consequence of intense, intermittent exposure of the skin to ultraviolet radiation, particularly in children, rather than from the cumulative lifetime exposure that is associated with other forms of skin cancer. Here we use a genetically engineered mouse model to show that a single dose of burning ultraviolet radiation to neonates, but not adults, is necessary and sufficient to induce tumours with high penetrance which are reminiscent of human melanoma. Our results provide experimental support for epidemiological evidence that childhood sunburn poses a significant risk of developing this potentially fatal disease.

Urocanic acid does not photobind to DNA in mice irradiated with immunosuppressive doses of UVB.

Ultraviolet B (UVB, 290-320 nm) radiation initiates in vivo a dose- and wavelength-dependent down regulation of cell-mediated immunity. An action spectrum for UV-induced immunosuppression indicated that the photoreceptor for this effect is urocanic acid (UCA), which undergoes a trans to cis isomerization in the stratum corneum on UV exposure. An accumulation of evidence has supported this conclusion. However, evidence has also been presented that formation of thymine dimers in DNA is responsible for initiation of UV-induced immunosuppression. Because photobinding of UCA to DNA in vitro forming cyclobutane-type adducts has been shown, we sought to resolve this dilemma by investigating if UCA photobinds to DNA in vivo. The [14C]cis-UCA, [14C]trans-UCA or [3H]8-MOP (8-methoxypsoralen) was applied topically to BALB/c mice that were then irradiated with a dose of UV previously shown to cause systemic suppression of contact hypersensitivity. The DNA was prepared from epidermal cells by phenol extraction immediately after in vivo irradiation and bound radioactivity determined. Although photobinding of [3H]8-MOP was readily demonstrable under these conditions (0.9 nmol/mg DNA), no significant binding of either isomer of UCA to DNA (between 1.2 x 10(-3) and 2.1 x 10(-3) ng/mg DNA) could be detected. Uptake studies in keratinocytes prepared from epidermis of untreated animals indicated that [3H]8-MOP was taken up with a rate constant of 4.2 x 10(-3) pmol/s/mg protein/mumol/L. In contrast, uptake of [14C]cis-UCA was not statistically significant from zero and uptake of [14C]trans-UCA was negligible (0.8 x 10(-3) +/- 0.08 x 10(-3) pmol/s/mg protein/mumol/L). There was no significant difference between uptake of UCA isomers, but uptake of [3H]8-MOP was significantly greater than that of either UCA isomer (P < 0.01). These studies indicate that the photobinding of UCA to DNA does not play a role in UV-induced immunosuppression.

Dietary L-histidine regulates murine skin levels of trans-urocanic acid, an immune-regulating photoreceptor, with an unanticipated modulation: potential relevance to skin cancer.

Solar ultraviolet-B radiation (UVB; 290-320 nm) causes skin cancer and suppresses cell-mediated immunity, preventing the rejection of UV-induced tumors. One mechanism initiating UV suppression involves the trans to cis photoisomerization of urocanic acid (UCA), a histidine derivative found in the stratum corneum. The addition of L-histidine to nonpurified mouse diet has been shown to increase skin trans-UCA levels and sensitivity to UVB immune suppression. Specially formulated L-histidine diets (0.40-64 g/kg) fed to BALB/c mice that were monitored over a 19-wk period resulted in an unexpected modulation of skin trans-UCA. ANOVA revealed a group-time interaction, providing initial evidence that the skin levels of trans-UCA were modulating up and down in all groups except the control group (6.4 g/kg diet). We observed that both high (64 g/kg diet) and low (0.4 g/kg diet) levels of dietary L-histidine resulted in the increase of skin trans-UCA to levels significantly higher than those recorded in the control group. In mice fed these histidine levels, skin trans-UCA increased to between 2.9 and 3.6 nmol/mg skin (64 g/kg diet, over 5 wk; 0.4 g/kg diet, over 8 wk) and then decreased to approximately 1.69 nmol/mg skin, the base-line level (64 g/kg diet, over 11 wk; 0.4 g/kg diet, over 17 wk). The increase in trans-UCA levels in mice with low L-histidine intake may be the result of protein malnutrition, consistent with weight loss observed in those mice. The modulation of trans-UCA levels in skin by dietary L-histidine has not been previously described; its role in skin cancer development is under investigation.

A low-barrier hydrogen bond in the catalytic triad of serine proteases? Theory versus experiment.

Cleland and Kreevoy recently advanced the idea that a special type of hydrogen bond (H-bond), termed a low-barrier hydrogen bond (LBHB), may account for the "missing" transition state stabilization underlying the catalytic power of many enzymes, and Frey et al. have proposed that the H-bond between aspartic acid 102 and histidine 57 in the catalytic triad of serine proteases is an example of a catalytically important LBHB. Experimental facts are here considered regarding the aspartic acid-histidine and cis-urocanic H-bonds that are inconsistent with fundamental tenets of the LBHB hypothesis. The inconsistencies between theory and experiment in these paradigm systems cast doubt on the existence of LBHBs, as currently defined, within enzyme active sites.

Microdialysis-immunoaffinity capillary electrophoresis studies on neuropeptide-induced lymphocyte secretion.

A micro-sampling procedure has been developed for studying lymphocyte secretion of biologically important peptides in low cell density cultures. The technique is based on microdialysis recovery of the analytes of interest coupled with immunoaffinity capillary electrophoresis separation of the microdialysis samples and laser-induced fluorescence detection. Although the technique is able to recover secreted materials only at the 5-10 cell level, the detection system has a limit of detection (LOD) in the attomole (10(-18) M) range. This degree of sensitivity indicates that the system has the potential to measure secreted products at the single cell level. An added advantage of this system over other sampling techniques is that the microdialysis probe allows continuous sampling over time.

The effects of UVA-I (340-400 nm), UVA-II (320-340 nm) and UVA-I+II on the photoisomerization of urocanic acid in vivo.

Ultraviolet B radiation (280-320 nm) can systemically suppress contact hypersensitivity (CHS), delayed type hypersensitivity (DTH) and tumor rejection responses in mice. Several models have been postulated for the initiation of this UVB-induced immune suppression and, although the complete mechanism is unclear, our early studies suggested that initiation is via the activation of a photoreceptor in the skin, identified as urocanic acid (UCA). Recent preliminary data from our laboratory and others indicated that UVA (320-400 nm)-emitting broad-band sunlamps can also isomerize UCA but may not lead to immune suppression, in contrast to UVB-emitting sunlamps, which cause both effects. Although the reason for this inconsistency is unknown, the emission spectra of UVA lamps contain differing amounts of UVB, UVA-I (340-400 nm) and UVA-II (320-340 nm) from those of UVB sources. In this study we determined a detailed dose-response for the isomerization of UCA in mouse skin using the UVA-I, UVA-II and UVA-I+II wavelength ranges. The dose-response curves obtained were put on an equal energy basis by quantum correction and the possibility of wavelength interaction for this effect investigated. A simple additive wavelength interaction between UVA-I, UVA-II, and UVA-I+II was observed for trans-UCA photoisomerization. This result indicates that the failure of UVA-I, UVA-II or UVA-I+II radiation to induce immune suppression of the CHS response in an animal model is not due to complex wavelength interactions and/or the presence of an in vivo endogenous photosensitizer of UCA isomerization. Other factors, such as downstream blocking by UVA of the cis-UCA generated signal, may be involved.

Dietary beta-carotene and ultraviolet-induced immunosuppression.

Ultraviolet (UV)-induced immunosuppression is a critical step in UV carcinogenesis, permitting tumour outgrowth. We investigated the effect of dietary beta-carotene on UV suppression of contact hypersensitivity (CHS) to trinitrochlorobenzene (TNCB) in BALB/c mice. Mice were fed for 10-16 weeks chow alone or supplemented with 1% beta-carotene or placebo as beadlets. Serum beta-carotene was detectable by high performance liquid chromatography (HPLC) analysis only in beta-carotene-fed mice (2.06 +/- 0.15 micrograms/ml). Serum retinol was 0.22-0.27 micrograms/ml in all three groups. Mice (n = 41/dietary group) were irradiated with 0, 4.5, 9 or 18 kJ/m2 of UVB and the CHS response was measured. Decreased CHS responses were observed in all UV-irradiated groups compared with unirradiated controls. UV dose-responses for suppression of CHS derived by first-order regression analyses of plots of percentage suppression of CHS as a function of log10UV dose showed significant slopes (P < 0.02) for all three dietary groups and similar residual variances between groups, P > 0.05. The UV dose for 50% suppression of CHS was 6.3 kJ/m2 for control, 6.4 kJ/m2 for placebo, and 5.5 kJ/m2 for beta-carotene-fed mice. No significant differences in slopes or elevations between UV dose-responses were observed, P > 0.05. Skin levels of the initiator of UV-induced immunosuppression, cis urocanic acid, were determined by HPLC in mice given 0 or 9 kJ/m2 of UV (n = 28/dietary group). No significant differences were observed between dietary groups (range 35.2-41.1 ng/mg skin, P > 0.15) We conclude feeding beta-carotene to BALB/c mice does not alter susceptibility to UV immune suppression, in contrast to human studies.

Immunosuppression by ultraviolet B radiation: initiation by urocanic acid.

Irradiation with UV-B, a component of natural sunlight, initiates systemic immunosuppression of delayed-type hypersensitivity responses. This may be a fundamental regulatory mechanism, controlling the interaction between mammals and potentially deleterious environmental UV radiation. Here, Frances Noonan and Edward De Fabo assess the evidence that suppression is initiated by the photoisomerization of trans-urocanic acid (UCA) in the stratum corneum, discuss the significance of this mechanism for skin cancer outgrowth and propose applications for UCA in transplantation.

cis-urocanic acid down-regulates the induction of adenosine 3',5'-cyclic monophosphate by either trans-urocanic acid or histamine in human dermal fibroblasts in vitro.

It has been demonstrated that UVB radiation (290-320 nm) suppresses mammalian cell-mediated immunity by effecting the trans to cis isomerization of urocanic acid (UCA) in the stratum corneum, the uppermost layer of the skin. Trans-urocanic acid has been shown to be the photoreceptor for UVB-induced immune suppression and the cis-isomer has been demonstrated to be immunosuppressive. Little is known, however, about how the isomerization of UCA may affect the proximal or distal cells of the skin or the immune system. We report here that trans-UCA is biologically active in vitro in human dermal fibroblasts, inducing adenyl cyclase as measured by cAMP (adenosine 3',5'-cyclic monophosphate) formation in a dose-dependent manner similar to the action of histamine. Trans-UCA and histamine stimulate 50% of maximum activity at concentrations of 3.3 microM and 13.8 microM respectively. Cis-UCA does not increase cAMP in these human fibroblasts but actively down regulates the increase of cAMP induced by either histamine or trans-UCA. Cis-UCA down regulated the histamine response by 75% and the trans-UCA response by 60% at a concentration range of 1 mM to 1 nM. The trans-UCA induction of cAMP can also be downregulated with an H2 histamine receptor antagonist cimetidine. These results support the hypothesis that a cellular target for cis-UCA is the dermal fibroblast and the effects reported here may represent the initial biochemical and cellular event for UVB-induced immune suppression i.e. the immediate step following the isomerization of trans to cis-UCA is the down regulation of cAMP by cis-UCA. Regulation of such an important second messenger such as cAMP could then allow cascading signals to occur, leading to immune suppression.

Dietary histidine increases mouse skin urocanic acid levels and enhances UVB-induced immune suppression of contact hypersensitivity.

Urocanic Acid (UCA) exists in mammalian skin primarily as the trans isomer and is photoisomerized to cis UCA upon UVB absorption. Our previous studies indicated that the photoisomerization of UCA is the initiating event in UBV-induced suppression of cell-mediated immunity (tUCA----cUCA----immune suppression). The purpose of this study was to verify the role of UCA in UV-induced immune suppression of contact hypersensitivity (CHS) in BALB/c mice. Since UCA is a metabolite of the amino acid L-histidine, we reasoned that increased dietary levels of histidine should raise skin tUCA levels. If skin tUCA is the UVB photoreceptor for immune suppression, this increase should enhance UV-induced suppression of CHS. HPLC analysis of skin from BALB/c mice given a histidine-rich diet (10%) showed that the total amount of UCA is significantly higher in these animals than in mice fed a normal diet. Further, levels of suppression of CHS of 3% and 49% in control fed mice, induced by 4.8 and 7.2 kJ/m2 UVB were significantly increased to 21% and 71% respectively in histidine-fed animals at these same UVB doses. These findings provide additional support for the UCA model for immune suppression, and provide the first evidence that UV-induced immune suppression can be enhanced by a dietary component, L-histidine.

Ultraviolet-B dose-response curves for local and systemic immunosuppression are identical.

Irradiation of mice with UVB suppresses contact hypersensitivity either "locally", i.e. when sensitizer is applied to the UV irradiated site, or "systemically", i.e. when sensitizer is applied to a site distal to the site of irradiation. It has been suggested that local suppression requires lower doses of UV than does systemic suppression, and that different mechanisms are therefore responsible. We undertook a detailed analysis of the dose-response and kinetics of UV-induced local and systemic suppression of contact hypersensitivity to trinitrochlorobenzene in two strains of mice, C57BL/6 and BALB/c. We found that the UV dose-responses for systemic and local suppression were identical within the same strain. Comparison, however, of UV dose-responses between strains indicated that C57BL/6 mice required 6.4 times less UV than did BALB/c mice to generate an equivalent amount of suppression. In both strains, local suppression was initiated if sensitizer was applied immediately, or 1 or 3 days after completion of a single dose of UV. In contrast, systemic suppression was initiated only if sensitizer was applied 3 days after UV irradiation. Thus local suppression was generated in the absence of significant systemic suppression (but not vice versa), and this was dependent on time of application of sensitizer after UV irradiation, not on the dose of UV administered. Filtration of the UV source with Mylar indicated that UVB was responsible for initiating both local and systemic suppression. In summary, these results indicate that (1) genetically determined differences in susceptibility to UV suppression exist, (2) the time courses of generation of local and systemic suppression are identical, and therefore use of the terms "low dose" and "high dose" to refer respectively to local and systemic suppression by UV irradiation are incorrect. We conclude that a common mechanism initiates UV-induced local and systemic suppression of contact hypersensitivity by the immediate formation, at the site of UV irradiation, of an immunosuppressive signal which takes between 1 and 3 days to act systemically.

Biologically effective doses of sunlight for immune suppression at various latitudes and their relationship to changes in stratospheric ozone.

Using information on solar irradiance at different latitudes derived from a radiative transfer model and a detailed in vivo action spectrum for immune suppression in a murine system, we report here calculations of the "biologically effective" irradiance of sunlight for immune suppression. From 40 degrees N to 40 degrees S in summer, under normal stratospheric ozone concentrations this value ranged from 0.27 W/m2 (40 degrees N or S) to a peak of 0.33 W/m2 (20 degrees N or S) predicting that 50% immune suppression in the Balb/c mouse would occur after 21-26 min of sunlight exposure within this latitude range. We also found that the most effective wavelengths for immune suppression shift from a peak of 270 nm in the laboratory to near 315 nm in sunlight. Furthermore, using ozone depletion scenarios of 5 to 20%, at latitudes 20 degrees S and 40 degrees N, a 0.6% increase in biologically effective irradiance levels of solar UVB for immune suppression was predicted for each 1% decrease of ozone. This value rose to a nearly 1% increase for each 1% decrease in ozone at 60 degrees N latitude in wintertime. These data indicate that activation of immune suppression, in a murine model, requires relatively low levels of sunlight and that these levels are easily obtainable over most of the populated regions of the world. Since a UVB-activated photoreceptor, urocanic acid, regulates immune suppression in mice and since this same compound exists on other mammalian skin, including human skin, suppression of the mammalian immune system is predicted to increase if substantial stratospheric ozone depletion takes place.

Cis-urocanic acid, a product formed by ultraviolet B irradiation of the skin, initiates an antigen presentation defect in splenic dendritic cells in vivo.

Urocanic acid (UCA, deaminated histidine) is a major ultraviolet-absorbing component of the stratum corneum. On UV irradiation, the naturally occurring trans form converts to the cis isomer. We have previously postulated that UV-induced systemic suppression is initiated by cis-UCA by way of an antigen-presenting cell defect. To test this hypothesis further, we have investigated the antigen-presenting cell (APC) function of splenic dendritic cells (DC). Splenic DC were prepared from mice 7 days after 1 h UV irradiation (27 kJ/m2) or i.v. administration of 50-200 micrograms/mouse of cis- or trans-UCA. Dendritic cells from UV-irradiated or cis-UCA-treated mice had a significantly impaired (APC) ability, assessed by the proliferative response of purified T cells from mice immune to DNP6 OVA to DC pulsed with this antigen. Dendritic cells from mice given trans-UCA had normal APC ability. The number of FcR+ cells was the same in DCs from all four treatment groups, and the number of IAd+ cells and the intensity of IAd expression were not decreased in DCs from UV-irradiated or cis-UCA-treated mice. Mixture of DCs from UV- or cis-UCA-treated mice with DCs from normal mice did not suppress APC activity. Dendritic cells taken 3 days after UV or cis-UCA treatment, in contrast to DC taken 7 days after treatment, had normal APC ability, indicating a time delay in the generation of the APC defect. In contrast, addition of cis-UCA or trans-UCA (66 micrograms/ml) directly to an in vitro proliferation assay had no effect, suggesting that cis-UCA may be activated in vivo. These results support our original hypothesis that cis-UCA has a natural role as a modulator of immune function.

Mechanism of systemic immune suppression by UV irradiation in vivo. II. The UV effects on number and morphology of epidermal Langerhans cells and the UV-induced suppression of contact hypersensitivity have different wavelength dependencies.

We previously reported that broad band UV radiation or narrow bands of UV (Hbw 3 nm) of wavelengths 250 to 320 nm cause a systemic suppression of contact hypersensitivity (CHS) in mice, observed when the contact sensitizer is applied to a nonirradiated site. To determine if this effect is associated with UV-induced alterations in epidermal Langerhans cell (LC) numbers and morphology, we performed the following study. LC were identified by ATPase staining of EDTA-separated epidermal sheets. Electron microscope studies confirmed that this method was a satisfactory indicator of the presence of LC; we found no evidence for LC which did not stain for ATPase in either irradiated or unirradiated epidermis. Mice were irradiated on the back with narrow band UV of peak wavelength 270, 290, or 320 nm. The irradiated skin was excised 24 hr later and was stained as described. The number of LC with ATPase staining dendrites and the number of nondendritic LC were enumerated. We found that UV radiation of 270 or 290 nm caused 1) an alteration in LC morphology (loss of dendrites) and 2) a decrease in the total number of epidermal LC. Both effects occurred in a dose-dependent fashion. Previously, these same wavelengths of narrow band UV, but at higher doses, had been shown to cause systemic suppression of CHS. In this study, the doses of 270 or 290 nm UV that resulted in the decreased LC numbers and alterations in LC morphology described above were insufficient to cause systemic suppression of CHS. The converse was found if the irradiating waveband of UV had a peak at 320 nm. A dose of 320 nm UV that caused 50% systemic suppression of CHS had no effect on either the number or the morphology of LC at the site of irradiation. In addition, the number and morphology of LC were unaffected in the ventral epidermis (site of contact sensitization) of mice that had been previously irradiated on the back with a systemically suppressive dose of UV. We conclude: (a) UV-induced alterations in the number and morphology of LC at the site of irradiation are not necessary for the generation of systemic suppression of CHS by UV radiation; this indicates that the initial UV-absorbing event triggering systemic suppression is neither a loss of, nor morphologic alterations to, LC at the irradiation site. (b) A systemic effect of UV radiation on the number and morphology of LC at the unirradiated site of contact sensitization does not occur, and thus is not responsible for the UV-induced systemic suppression of CHS by UV radiation.

Mechanism of immune suppression by ultraviolet irradiation in vivo. I. Evidence for the existence of a unique photoreceptor in skin and its role in photoimmunology.

UV irradiation of mice causes a systemic immune alteration that can be detected either by suppression of the immunologic rejection of UV-induced tumors, or by suppression of contact hypersensitivity (CHS). Suppression of these two immunologic responses has similar photobiologic characteristics and in both cases is associated with the generation of antigen-specific suppressor T cells. To identify whether a specific photoreceptor for this effect exists, the relative wavelength effectiveness (action spectrum) was determined for the UV-induced suppression of CHS. Narrow bands of UV (half bandwidth 3 nm) were used at 10 wavelengths from 250 to 320 nm to obtain dose-response curves. Irradiation with each of these bands of UV caused dose-dependent immunosuppression of CHS, but with differing effectiveness. Immunosuppression was clearly separable from the generation of gross skin damage and inflammation. Further, immunosuppression by the most effective wavelength (270 nm) was associated with the generation of antigen-specific suppressor cells. The action spectrum derived from the dose-response curves has a maximum between 260 and 270 nm, a shoulder at 280-290 nm, and declines steadily to approximately 3% of maximum at 320 nm. The finding of such a clearly defined wavelength dependence implies the presence of a specific photoreceptor for this effect. Removing the stratum corneum by tape stripping before UV irradiation prevented the suppression of CHS using 254-nm radiation, suggesting the photoreceptor is superficially located in the skin. A number of epidermal compounds with absorption spectra similar to the action spectrum are discussed and evaluated with respect to their potential for being the photoreceptor. Based on (a) the close fit of its absorption spectrum to the action spectrum, (b) its superficial location in the stratum corneum, and (c) its photochemical properties, the hypothesis is advanced that the photoreceptor for systemic UV-induced immunosuppression of contact hypersensitivity may be urocanic acid. As such, it may also play a role in UV-induced carcinogenesis via the production of tumor-specific suppressor cells.